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  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
  • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
  • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams
  • C07D205/085—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams with a nitrogen atom directly attached in position 3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
  • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
  • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams
  • C07D205/09—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams with a sulfur atom directly attached in position 4
  • C07D205/095—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams with a sulfur atom directly attached in position 4 and with a nitrogen atom directly attached in position 3
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

• This invention relates to novel compounds, pharmaceutical compositions thereof, and their use as anti-inflammatory agents in mammals.
• Coenzyme A-independent transacylase is an enzyme responsible for the movement of arachidonate between phospholipid molecular species of inflammatory cells. CoA-IT removes arachidonate from the sn-2 position of 1-acyl- containing phospholipids, such as l-acyl-2-arachidonoyl-sn-glycero-3- phosphocholine ( l-acyl-2-arachidonoyl-GPC).
• lyso-phospholipid acceptor such as l-alkyl-2-lyso-GPC and l-alkenyl-2- lyso-sn-glycero-3-phospho-ethanolamine (Sugiura et al., J. Biol. Chem. 262: 1 199- 1205 (1987); Kramer and Deykin, Biol. Chem. 258: 13806- 13811 (1983); Chilton et al., J. Biol. Chem. 258: 7268-7271 ( 1983)).
• a suitable lyso-phospholipid acceptor such as l-alkyl-2-lyso-GPC and l-alkenyl-2- lyso-sn-glycero-3-phospho-ethanolamine
• This activity is selective for 20 carbon fatty acyl groups and is the mechanism by which inflammatory cells move arachidonate into specific phospholipid pools prior to its release (Winkler and Chilton, Drug News Perspec. 6: 133-138 (1993); Snyder et al., J. Lipid Mediat. 10: 25-31 (1994)). Further, a method which antagonises the production of free arachidonic acid, its metabolites or PAF will have clinical utility in the treatment of a variety of allergic, inflammatory and hypersecretory conditions such as asthma, arthritis, rhinitis, bronchitis and urticaria, as well as reperfusion injury and other disease involving lipid mediators of inflammation.
• CoA-IT is involved in arachidonic acid and phospholipid metabolism
• inhibition of such an enzyme would be useful for the treatment of inflammatory, allergic and hypersecretory conditions or disease states caused thereby. Therefore, a method by which CoA-IT is inhibited will consequently and preferentially decrease the arachidonate content of 1-alkyl- and 1-alkenyl-linked phospholipids and will therefore decrease the production of pro-inflammatory mediators such as free arachidonic acid, prostaglandins, leukotriene and PAF during " an inflammatory response.
• pro-inflammatory mediators such as free arachidonic acid, prostaglandins, leukotriene and PAF
• CoA-IT inhibitors i.e. compounds which are capable of inhibiting, or interfering with this enzyme and thereby decrease production of the pro-inflammatory mediators.
• This invention also relates to a method of treating or reducing inflammation in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound or composition of Formula (I).
• This invention also relates to a method of treating disease or disorders mediated by lipid inflammatory mediators, free arachidonic acid, its metabolites and/or PAF by administering to a patient in need thereof, an effective amount of a compound of Formula (I).
• This invention also relates to a method of treating disease or disorders mediated by Coenzyme A independent transacylase (CoA-IT) by administering to a patient in need thereof, an effective amount of a compound or composition of Formula (I).
• CoA-IT Coenzyme A independent transacylase
• This invention relates to the novel compounds of Formula (la) and pharmaceutically acceptable salts thereof.
• the present invention also provides for a pharmaceutical composition comprising a pharmaceutical acceptable carrier or diluent and a compound of Formula (la), or pharmaceutically acceptable salt thereof.
• One aspect of the present invention are the compounds represented by a structure having the formula:
• Y is NH
• X is O or S(O)m; m is 0 or an integer having a value of 1 , or 2; R3 is optionally substituted triphenylmethyl; R4 is optionally substituted C J . or
• n is an integer having a value of 1 to 4;
• RlO and R20 are independently hydrogen or C 1.4 alkyl;
• R5 is hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, O ⁇ R ⁇ , or
• R5 is C ⁇ _ ⁇ o alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl, all of which may be optionally substituted
• R7 is independently hydrogen, Cj.io alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl, all of which may be optionally substituted; or a pharmaceutically acceptable salt thereof.
• Figure 1 demonstrates the time dependent inhibition of CoA-IT by (3S,4R)-4-
• Figure 2 demonstrates inhibition of PAF production in neutrophils by (3S,4R)-4-
• the present invention is directed to a novel method of treating inflammatory disease in a mammal in need thereof by administering to said mammal an effective amount of a compound according to Formula (I).
• the compounds of Formula (I) selectively inhibit the CoA-IT enzyme. This will result in the treatment of inflammatory occurrences in mammals.
• Inflammatory states in mammals may include, but are not limited to, allergic and asthmatic manifestations, dermatological diseases, inflammatory diseases, collagen diseases, reperfusion injury and stroke. Treatment of both acute and chronic diseases are possible.
• Preferred diseases for treatment are arthritis, asthma, allergic rhinitis, inflammatory bowel disease (IBD), psoriasis, reperfusion injury and stroke.
• the compounds of Formula (I) are preferential and selective inhibitors of CoA-IT.
• X is O or S(O)m; and m is 0 or an integer having a value of 1 , or 2.
• m is 0 or 2.
• R3 is optionally substituted triphenylmethyl group.
• the phenyl rings may be independently substituted one to three times by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy substituted Ci-ioalkyl; Ci-io alkoxy, such as methoxy or ethoxy; halosubstituted C j- io alkoxy; S(O)m alkyl, such as methyl thio, methylsulfinyl or methyl sulfonyl; S(O)m aryl; amino, mono & di- C i-io alkyl substituted amino;
• Ci-io alkyl halosubstituted Ci-io alkyl, such as CF3; CHO, C(O)C ⁇ _ ⁇ o alkyl, C(O) aryl, C(O)2Rg, wherein Rg is C ⁇ _ ⁇ o alkyl, aryl, or arylalkyl; C(O)NR9R 1 , cyano; S(O) 2 NR 9 R ⁇ ; N(R 10 )C(O)R 6 ; N(R 10 )C(O) NR 9 R ⁇ ; N(R 10 )C(O)OR 6 ; or N(R 10 )S(O) 2 R6.
• R9 and Rj 1 are independently hydrogen, C ⁇ IQ alkyl, aryl, arylalkyl.
• R4 is optionally substituted C ⁇ . ⁇ Q alkyl
• n is an integer having a value of 1 to 4.
• n is 1.
• R4 is an optionally substituted C ⁇ . ⁇ Q alkyl
• the alkyl moiety may be straight or branched, and may be substituted one or more times, independently by halogen, such as fluorine; hydroxy; Ci-io alkoxy; S(O)m alkyl, wherein m is 0, 1 or 2; amino, mono & di-substituted amino, such as NR9R1 1 group; wherein R9 and Rl 1 are as described above, or R9 and Ri 1 together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from O/N/S; -O(CR ⁇ oR*2 ⁇ )sO- wherein s is an integer having a value of 2 to 4 and both oxygens are attached to the same carbon in R4; - S(CRi () 2 ⁇ )sS ⁇ wherein s is as previously defined and both sulfurs are attached to the same carbon in R4; cycloalky
• R4 is a ⁇ . ⁇ . alkyl, such as isobutyl, or an alkenyl, such as isobutenyl.
• R JQ and R20 are independently hydrogen or C 1.4 alkyl.
• R5 is hydrogen, C ⁇ J Q alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, C(O)2R ⁇ , C(O)R6-
• R5 is hydrogen, C(O)2R6, or a heteroaryl ring, and preferably R therein is a C 1.4 alkyl, such as methyl.
• R5 is a heteroaryl ring, it is preferably a 2-, 3,- or 4-pyridyl.
• the alkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl may be optionally substituted as herein defined.
• R is _ I alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl.
• the aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, and heterocyclicalkyl moieties may be optionally substituted as herein defined.
• R7 is independently hydrogen, C ⁇ _ Q alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl.
• the alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, and heterocyclicalkyl moieties may be optionally substituted as herein defined.
• Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid
• basic salts of inorganic and organic acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and man
• halo all halogens, that is chloro, fluoro, bromo and iodo;
• cycloalkyl is used herein to mean cyclic radicals, preferably of 3 to 8 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like;
• alkenyl is used herein at all occurrences to mean straight or branched chain radical of 2-10 carbon atoms, unless the chain length is limited thereto, including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl and the like;
• heteroaryl alkyl - a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole, or benzimidazole; • "heterocyclic” (on its own or in any combination, such as
• heterocyclylalkyl a saturated or partially unsaturated 4-10 membered ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O, or S; such as, but not limited to, pyrrolidine, piperidine, piperazine, morpholine, tetrahydropyran, or imidazolidine; •
• aralkyl or “heteroarylalkyl” or “heterocyclicalkyl” is used herein to mean Cj-4 alkyl as defined above attached to an aryl, heteroaryl or heterocyclic moiety as also defined herein unless otherwise indicate;
• alkanoyl - a C(O)Ci-io alkyl wherein the alkyl is as defined above.
• optionally substituted shall mean such groups as halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy substituted Ci-ioalkyl; Ci-io alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; amino, mono & di-substituted amino, such as in NR9R11 group; Ci-io alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc., cyclopropyl, or cyclopropyl, or cyclopropyl
• the compounds of the present invention may exist as stereoisomers, regioisomers, or diastereoisomers. These compounds may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds are included within the scope of the present invention.
• Another aspect of the present invention are the novel compounds of Formula (la) represented by the structure:
• Y is NH
• X is O or S(O)m; m is 0 or an integer having a value of 1, or 2;
• R 3 i optionally substituted triphenylmethyl
• R4 i optionally substituted C io alkyl, or
• R ⁇ O and R20 are independently hydrogen or Cj_4 alkyl
• R5 is hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, C(O)2R6 > or
• Rg is Cj_ ⁇ alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl, wherein the alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl moieties may be optionally substituted;
• R7 is independently hydrogen, C ⁇ _ ⁇ o alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, or heterocyclicalkyl, wherein the alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic, and heterocyclicalkyl moieties may be optionally substituted; excluding 4-methoxy-3-(triphenylmethylamino)azetidin-2-one, 3- (triphenylmethylamino)-azetidin-2-one, 4-(isobutenyloxy)-3-
• Specifically exemplified compounds of Formula (I) are: (3RS,4RS)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one (3R,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one (3S,4S)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one (3R,4R)-4-(Isobutylthio)-3-(triphenylmethylamino)azetidin-2-one (3R,4R)-4-(Isobutylsulfonyl)-3-(triphenylmethylamino)azetidin-2-one (3S,4R)-4-(Isobutoxy)-3-(triphenylmethylamino)azetidin-2-one (3S,4R)-4-(Isobutoxy)-3-(tri
• the displacement may be carried out in the presence of a suitable base, such as aqueous sodium hydroxide, in a suitable solvent such as acetone.
• a suitable base such as aqueous sodium hydroxide
• a suitable solvent such as acetone.
• Compound 3 where 4-(X-R 4 ) is 4JSO-R 4 ) and 4-(SO2-R 4 ) and R3, R 4 are defined as in formula (I) can be obtained by further oxidation of 2 where 4-(X-R 4 ) is 4-(S-R 4 ) with a suitable organic oxidizing agent such as m-chloroperbenzoic, peracetic acid, etc. in a suitable solvent such as dichloromethane, or by further oxidation with a suitable inorganic oxidizing agent such as sodium periodate or potassium permangante in a solvent such as water, acetone or acetic acid.
• a suitable organic oxidizing agent such as m-chloroperbenzoic, peracetic acid, etc
• compounds of formula (I) may be prepared according to Scheme II from 4.
• Compound 4, wherein X is O-R 4 or S-R 4 and R 4 is as defined in formula (I) and Rl is hydrogen, can be prepared from 4-acetoxy or 4-benzoyloxy- azetidin-2-one as described in Synthetic Communications, 24, 131-135 (1994) whose disclosure is incorporated herein by reference.
• Treatment of 4, where R* is hydrogen, with a suitable silylating group such as tert-butyldimethylsilyl chloride and a suitable base such as triethylamine in a suitable solvent such as tetrahydrofuran gives 4 where ⁇ is tert-butyldimethylsilyl.
• Rl is tert-butyldimethylsilyl
• a suitable base such as lithium diisopropylamide in a suitable solvent such as tetrahydrofuran at a suitable temperature such as -50°C
• a suitable azidating reagent such as tosyl azide in a suitable solvent such as tetrahydrofuran
• trimethylsilyl chloride gives 5 where R ⁇ is tert-butyldimethylsilyl.
• compounds II-5 may be prepared using [2 + 2] cycloaddition reactions, for example, by following the general procedures described in Cama et. al., Tetrahedron Letters, 4233, 1978, whose disclosure is incorporated herein by reference.
• reaction mixture was stirred for 40 min, the temperature was lowered to -70°C, and the mixture was treated dropwise over 10 min with a solution of l-(tert-butyldimethylsilyl)-4-(isobutoxy)-azetidin-2-one (1.8 g, 7 mmol) in tetrahydrofuran (7 mL).
• the mixture was stirred for 1 h, transferred to a jacketed addition funnel maintained at -70°C, and added over 40 min to a solution of p-toluenesulfonyl azide (1.8 g, 9 mmol) in tetrahydrofuran (8 mL) containing hexamethylphosphoramide (2 mL) maintained at -70°C.
• the reaction was stirred for 1 h and at -50°C for 4.5 h.
• the mixture was stirred at -28°C for 16 h, and trimethylsilyl chloride (5 mL) was added and the mixture was stirred at RT for 45 min.
• the mixture was diluted with water and extracted with ethyl acetate.
• Example 8 (3S,4S)-4-(Propoxy)-3-(triphenylmethylamino)azetidin-2-one: MS(ES) m/e 773 [2M+H]+;
• Example 9 (3S.4R)-4-(Benzyloxy)-3-(triphenylmethylamino)azetidin-2-one:
• Example 11 (3S,4R)-4-Methoxy-3-(triphenylmethylamino)azetidin-2-one: MS(ES) m/e 359 [M+H]+;
• Example 12 (3S,4R)-4-(Isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one:
• Examples 18-21 The following compounds were prepared using the general procedure of Example 3, except substituting: Examples 18 and 19: methyl 4-hydroxy-2-butynoate (Zh. Obshch. Khim. 66, 106, 1996), Example 20: 4-hydroxymefhyl-2(5H)-furanone, (J. Chem. Res., Synop. 222, 1986), or
• Example 22 (3S,4S)-4-(Prop-2-vnyloxy)-3-(triphenylmethylamio)azetidin-2-one: J. Chem. Soc. Perkin Trans. I 2268, 1979;
• Example 26 Using analagous procedures to those noted therein, the racemic mixture of (RS)-3-(Triphenylmethylamino)azetidin-2-one may be produced.
• the compounds of Formula (I) or pharmaceutically acceptable salts thereof can be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of an inflammatory disease state in a mammal, preferably a human.
• Inhibition of CoA-IT and the simultaneous reduction of PAF, free arachidonic acid and eicosanoid release from inflammatory cells according to this invention is of therapeutic benefit in a broad range of diseases or disorders.
• the invention herein is therefore useful to treat such disease states both in humans and in other mammals.
• Inhibition of CoA-IT by the compounds of Formula (I) is an effective means for simultaneously reducing PAF, free arachidonic acid and eicosanoids produced in inflammatory cells.
• the therapeutic utility of blocking lipid mediator generation has been recognized for many years.
• inhibitors of cyclooxygenase such as aspirin, indomethacin, acetaminophen and ibuprofen, have demonstrated broad therapeutic utilities.
• CoA-IT inhibitors inhibit cyclooxygenase products.
• Another class of inhibitors which are used in a broad range of inflammatory disorders are the corticosteroids. Corticosteroids act in a variety of ways, e.g.
• Both 14 kDa PLA2 inhibitors and CoA-IT inhibitors block the release of free arachidonic acid.
• Inhibitors of 5- hpoxygenase block the production of leukotrienes and leukotriene antagonists prevent the bioactions of leukotrienes. Recent studies indicate that both will have broad therapeutic utilities.
• Both 14 kDa PLA2 inhibitors and CoA-IT inhibitors block the production of leukotrienes.
• Inhibitors of phospholipase A2 block the release of free arachidonic acid and the formation of lyso PAF (the immediate precursor of PAF). PLA2 inhibitors are recognized to have broad therapeutic utilities.
• PAF Intravenous infusion of PAF at doses of 20-200 pmol kg ⁇ - 1 > min ⁇ - 1 > into rats has been reported to result in the formation of extensive haemorrhagic erosions in the gastric mucosa.
• Psoriasis is an inflammatory and proliferative disease characterised by skin lesions.
• PAF is pro-inflammatory and has been isolated from lesioned scale of psoriatic patients indicating PAF has a role is the disease of psoriasis.
• increasing evidence supports a potential patho-physiological role for PAF in cardiovascular disease.
• Treatment of disease states caused by these lipid inflammatory mediators i.e., arachidonate, eicosanoids and PAF include certain cardiovascular disorders such as but not limited to, myocardial infarction, stroke, circulatory shock, or hypotension, ischemia, reperfusion injury; inflammatory diseases such as, but not limited to, arthritis, inflammatory bowel disease, Crohn's disease, or ulcerative colitis; respiratory diseases such as but not limited to, asthma, or adult respiratory distress syndrome; analphylaxis, shock, such as but not limited to endotoxic shock; topical disesases, such as but not limited to actinic keratosis, psoriasis, or contact dermatitis; or pyresis.
• cardiovascular disorders such as but not limited to, myocardial infarction, stroke, circulatory shock, or hypotension, ischemia, reperfusion injury
• inflammatory diseases such as, but not limited to, arthritis, inflammatory bowel disease, Crohn's disease, or ulcerative colitis
• a compound of formula (I) or a pharmaceutically acceptable salt thereof in therapy it will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice.
• This invention also relates to a pharmaceutical composition comprising an effective, non-toxic amount of a compound of formula (I) and a pharmaceutically acceptable carrier or diluent.
• Compounds of formula (I), pharmaceutically acceptable salts thereof and pharmaceutical compositions incorporating such may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
• the compounds of formula (I) may be administered in conventional dosage forms prepared by combining a compound of formula (I) with standard pharmaceutical carriers according to conventional procedures.
• Such pharmaceutically acceptable carriers or diluents and methods of making are well known to those of skill in the art, and reference can be found in such texts as Remington's Pharmaceutical Sciences, 18th Ed., Alfonso R. Genarao, Ed., 1990, Mack Publishing Co. and the Handbook of Pharmaceutical Excipents, APhA Publications, 1986.
• the compounds of formula (I) may also be administered in conventional dosages in combination with known second therapeutically active compounds, such as steroids or NSAID's for instance. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
• the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• the pharmaceutical carrier employed may be, for example, either a solid or liquid.
• Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
• Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like.
• the carrier or diluent may include time delay material well known to the art, such as " glyceryl mono-stearate or glyceryl distearate alone or with a wax.
• a wide variety of pharmaceutical forms can be employed.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but preferably will be from about 25mg. to about lg.
• the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
• Compounds of formula (I) may be administered topically, that is by non- systemic administration. This includes the application of a compound of formula (I) externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream.
• systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
• Formulations suitable for topical administration include liquid or semi- liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
• the active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, for instance from 1 % to 2% by weight of the formulation. It may however comprise as much as 10% w/w but preferably will comprise less than 5% w/w, more preferably from 0.1% to 1 % w/w of the formulation.
• Lotions according to the present invention include those suitable for application to the skin or eye.
• An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
• Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
• Creams, ointments or pastes according to the present invention are semi- solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base.
• the base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel.
• the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan esteror a polyoxyethylene derivative thereof.
• Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
• Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent.
• the resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100 °C. for half an hour.
• the solution may be sterilized by filtration and transferred to the container by an aseptic technique.
• bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).
• Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
• Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of a compound of the structure (I) or a pharmaceutically acceptable salt thereof calculated as the free base.
• the pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen.
• a daily dosage regimen for an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered from 1 to 4 times per day.
• the choice of form for administration, as well as effective dosages will vary depending, inter alia, on the condition being treated. The choice of mode of administration and dosage is within the skill of the art.
• various cellular assays can be used to determine in vitro activity.
• various classical in vivo acute inflammatory models which have some aspect of their etilogy to elevated eicosanoid levels can be employed, such as the paw edema model, mouse zymosan peritonitis, reverse Arthus pleurisy or various skin inflammation assays which are described in Lewis et al., Experimental Models of Inflammation, in the Handbook of Inflammation, Vol. 5, Bonta Ed., Elsevier Science Publishers, NY (1985) whose disclosure is herein incorporated by reference.
• the TPA induced ear edema model is described herein as well.
• Inflammatory responses are induced in the mouse ear by the topical application of a pro-inflammatory agent, such as 12-O-tetradecanoyl- phorbol 13-acetate. This produces an edematous response, as measured by increases in ear thickness, as well as increased inflammatory cellular infiltrate, as measured by increases in myeloperoxidase activity (as described in the methods).
• a pro-inflammatory agent such as 12-O-tetradecanoyl- phorbol 13-acetate.
• This produces an edematous response, as measured by increases in ear thickness, as well as increased inflammatory cellular infiltrate, as measured by increases in myeloperoxidase activity (as described in the methods).
• inflammation induced by the direct adminstration of arachidonic acid can be used. In this case compounds altering arachidonic acid mobilization or liberation should be with our effect.
• the following is a method to measure CoA-IT activity and the effects of compounds on CoA-IT activity.
• the assay is based upon mixing cellular material containing CoA-IT activity with a stable lyso phospholipid such as l-alkyl-2-acyl- GPC and measuring the production of phospholipid product such as l-alkyl-2-acyl- GPC occurring in the absence of added CoA or CoA-fatty acids.
• Any inflammatory cell that contains high levels of CoA-IT activity can be used, such as neutrophils, macrophages or cell lines such as U937 cells.
• U937 cells were obtained from American Type Culture Collection and grown in RPMI-1640 media (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) at 37°C, 5%C02- Cells were grown without differentiation
• inflammatory cells include, but are not limited to neutrophils, macrophages, monocytes, lymphocytes, eosinophils, basophils, and mast cells.
• Microsomes were prepared using standard techniques. In this case, cells were washed with a buffer of 250 mM sucrose, 10 mM Tris, 1 mM EGTA, 1 mM MgCl2, pH 7.4 and ruptured by N2 cavitation (750 psi, 10 minutes). The ruptured cells were centrifuged 1000 X g, 5 minutes. The resulting supernatant was centrifuged at 20,000 X g, ⁇ 20 minutes. Microsomes were prepared from this supernatant by centrifugation at 100,000 x g, 60 minutes.
• the resulting pellet was washed once with assay buffer (150 mM NaC 1 , 10 mM Na2KPO4, 1 mM EGTA, pH 7.4), recentrifuged and the pellet resuspended in assay buffer (4-20 mg protein/ml) and was stored at -80°C until assayed.
• assay buffer 150 mM NaC 1 , 10 mM Na2KPO4, 1 mM EGTA, pH 7.4
• CoA-IT activity was measured in 1.5 ml centrifuge tubes in a total volume of 100 ul. Microsomes were diluted in assay buffer to the desired protein concentration (6-20 ug/tube). The reaction was initiated by addition of [-1H ]l-alkyl- 2-lyso-sn-glycero-3-phosphocholine (GPC) ( ⁇ 0.1 uCi/tube) and 1 ⁇ M final cold 1- alkyl-2-lyso-GPC in assay buffer with 0.25 mg/ml fatty acid-poor bovine serum albumin (BSA) (Calbiochem, La Jolla, CA).
• GPC [-1H ]l-alkyl- 2-lyso-sn-glycero-3-phosphocholine
• BSA bovine serum albumin
• [ 3 H] l-alkyl-2-lyso-GPC approximately 50 Ci/mmol, was from NEN-Dupont (Boston, Massachusetts) and cold l-alkyl-2-lyso-GPC was from Biomol (Plymouth Meeting, Pennsylvania).
• Microsomes were pretreated with desired agents for the desired time (10 minutes) before the addition of [3H]l-alkyl-2-lyso-GPC.
• the reaction was run for the desired time (10 minutes) at 37°C.
• the reaction was stopped and the lipids extracted by addition of 100 ul of chloroform: methanol ( 1 :2, v/v) followed by 100 ul of chloroform and 100 ul of 1 M KC1.
• Protein concentration were assessed using the protein assay reagents from Bio-Rad (Richmond, California).
• 5-LO 5- lipoxygenase
• CO cyclooxygenase
• the anti-oxidant BHT also has no effect at concentrations up to 100 ⁇ M.
• Compounds which complex with phospholipids and inhibit PLA2 activity, such as quinacrine and aristolochic acid have no effect on CoA-IT activity at concentrations up to 500 ⁇ M.
• Doxepine a compound reported to inhibit PAF release did not inhibit CoA-IT at concentrations up to 100 ⁇ M.
• Sodium diclofenac reported to decrease leukotriene production by altering arachidonic acid metabolism, had no effect on CoA-IT activity at concentrations up to 500 ⁇ M.
• Figure 1 demonstrates the inhibition of CoA-IT activity in a time dependent manner using a representative compound of Formula (I), Example 24, (3S,4R)-4- (isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one.
• Other representative compounds of Formula (I) which inhibited CoA-IT activity, as a time-dependent inhibitor, in the microsomal CoA-IT assay described above are:
• Human neutrophils are obtained in the laboratory using three different methods. One method uses leukophoresis packs from normal humans and neutrophils are isolated using the histopaque-1077 technique. The blood is centrifuged at 300 x g for 10 minutes. The cell pellets are resuspended in PBS composed of 137 mM NaCl, 8.8 mM Na 2 HPO 4 , 1.5 mM KH2PO4, 2.1 mM KC1
• the pellets are collected after centrifugation (300 x g for 30 minutes) and washed once in PBS. The cell pellets are exposed briefly to deionized water to lyse any erythrocytes. The remaining cells are collected by centrifugation, suspended in PBS, counted and identified after cytospinning and staining. The final leukocyte preparation will be of greater than 95% purity and viability.
• the second method isolates human neutrophils from fresh heparinized normal blood using the Histopaque-1077 technique.
• the blood is layered over Histopaque-1077 (Sigma, St. Louis Missouri) and centrifuged at 400 x g for 30 minutes.
• the cell pellets are resuspended in 35 ml of PBS and 12 ml of 6% Dextran, followed by Dextran sedimentation at room temperature for 45 minutes.
• the upper layer is collected and further centrifugated for 10 minutes at 1000 rpm.
• the cell pellets are exposed briefly to deionized water to lyse erythrocytes.
• the remaining cells are collected by centrifugation, suspended in PBS, counted and identified after cytospinning and staining.
• the final leukocyte preparation will be of greater than 95% purity and viability.
• the third method isolates human neutrophils from freshly drawn heparinized normal blood using the Percoll technique.
• the blood is first treated with 6%
• Dextran at room temperature for a 1 hour sedmination The upper layers of plasma are collected and centrifuged at 400 x g for 10 minutes.
• the cell pellets are resuspended in Percoll 1.070 g/ml supplemented with 5% fetal bovine serumand layered on discontinuous gradients (1.080, 1.085, 1.090,1.095 g/ml) followed by centrifugation at 400 x g for 45 minutes.
• the neutrophils are collected from interfaces of 1;080 and 1.085 and the 1.085 and 1.090 Percoll densities, followed by a centrifugation at 400 x g for 45 minutes.
• the neutrophils are suspended in PBS, counted and identified after cytospinning and staining.
• the final leukocyte preparation will be of greater than 95% purity and viability. There should be no difference noted in the response of the neutrophils nor in the effects of test compounds in neutrophils isolated by the three different techniques.
• Neutrophils were suspended in PBS at concentrations of 5 to 20 x 10" cells per ml. Cells were added to test tubes and treated with the desired compounds for 5 to 10 minutes, then challenged with calcium ionophore A23187, 2 ⁇ M and 20-30 ⁇ Ci of [*-1H]acetic acid (NEN-Dupont, Boston, Massachusetts), or the vehicle of PBS with 0.25-1 mg/ml. After 5 to 20 minutes, the reactions were terminated by addition of an equal volume of chloroform: methanol (1:2, v/v) to the samples and the lipids were extracted by addition of equal volumes of chloroform and distilled water. The samples were vortexed and centrifuged at high speed and the chloroform layer removed to a clean tube.
• the chloroform from each tube was evaporated to dryness and the material suspended in a small volume of chloroform or chloroform: methanol (25-100 ⁇ l) and the total material spotted on a Silica TLC plate.
• the plates were developed in chloroform/methanol/ acetic acid/water (50:25:8:4, v/v) visualized by radioscanning (Bioscan) and the product, [3H]PAF, was scraped and quantified by liquid scintillation spectroscopy.
• the Rf value for a synthetic standard of PAF is approximately 0.33.
• Figure 2 demonstrates that a representative compound of Formula 1, Example 24 (3S,4R)-4-(Isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one blocks PAF production in neutrophils.
• Leukocyte-rich leukopaks obtained from Biological Specialties (Lansdale, PA) were collected from male volunteers who were not taking anti-inflammatory drugs. Leukopaks were centrifuged (90 x g for 15 min) twice to remove the platelet-rich plasma. The cell pellet was washed by centrifugation and resuspended in HBSS without Ca 2"1" or Mg 2+ . Histopaque 1077 was layered under the cell suspension and centrifuged at 400 x g for 30 min to obtain the buffy coat. The interfacial buffy coat, containing monocytes and lymphocytes, was removed and saved.
• the buffy coat was washed twice with HBSS without Ca 2+ or Mg 2+ by centrifugation.
• the cell pellet (4-6 x 108 cells/30mls) was resuspended in iso-osmotic media (RPMI-1640, 10% heat inactivated fetal bovine serum (FBS), 0.2 mM L-glutamine, 2.5 mM HEPES) and layered over an equal volume of 46% Percol mixture (10X PBS/ Percol; 9.25 / 0.75) and 54% iso-osmotic media and centrifuged for 30 min at 1000 x g (Marshall and Roshak, Biochem. Cell Biol. 71: 331-339, 1993).
• the monocyte population located at the interface of the Percoll gradient was removed and washed twice in HBSS without Ca 2+ or Mg 2+ . This resulted in a greater than 85-90 % pure monocyte population as assessed by differential staining.
• Monocytes (5 x l ⁇ 6/ml) were incubated as a suspension in serum- free RPMI-1640 medium containing the vehicle DMSO ( ⁇ 1%) or drug for 30 min at 27°C after which vehicle or stimuli was added for the indicated time.
• the stimulating agent is solubilized in DMSO and appropriate vehicle controls were included in all experiments.
• the amount of stimuli was chosen from the linear portion of a concentration versus product curve usually representing 60-80% maximal stimulation over the indicated incubation time at 37°C (A23187, 1 ⁇ M, 15 min).
• the reaction was terminated by reduction of pH through addition of citric acid and centrifugation (10 min, 400 x g, 4°C).
• Figure 3 demonstrates that a representative compound of Formula (I), Example 24, (3S,4R)-4-(isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one, Example 24 demonstrated positive activity in this assay.
• the Figure shows that this compound through its CoA-IT inhibition blocked the production of leukotriene and prostaglandin, and that such inhibition was dependent upon the pre-treatment time.
• mice Male Balb/c inbred mice were obtained from Charle River Breeding Laboratories (Kingston, NY). Within a single experiment mice (22-25g) were age- matched. These in vivo experiments typically involved use of 5-6 animals/group.
• TPA (12-O-tetradecanoylphorbol 13-acetate) (Sigma Chemical Company) in acetone (4 mg/20ml) was applied to the inner and outer surfaces of the left ear of BALB/c male mice.
• the thickness of both ears was then measured with a dial micrometer (Mitutoyo, Japan) at both 2 and 4 hours after treatment, and the data expressed as the change in thickness (lO ' ⁇ cm) between treated and untreated ears.
• the application of acetone did not cause an edematous response; therefore, the difference in ear thickness represented the response to the TPA.
• the inflammed left ears were removed and stored at -70°C until they were assayed for MPO (myeloperoxidase) activity where appropriate.
• MPO myeloperoxidase
• MPO Mveloperoxidase
• ED50 are values which cause a 50% inhibition of the inflammatory response and are calculated by regression analysis of the dose response data. As shown below in Table 1, a representative compound of Formula (I), (3S,4R)-4-(isobutenyloxy)-3-(triphenylmethylamino)azetidin-2-one, Example 24 demonstrated positive activity in this assay.
• mice were given PMA followed by the test compound .
• the edematous response was measured using a thickness gauge 4 hrs post PMA application.
• the animals were sacrificed and the inflamed ears harvested and MPO extracted and assayed spectrophotometrically as described in methods. Both compounds produced effects significantly different from vehicle treatment.
• the positive activity of the compounds of Formula (I) in this animal model demonstrate a clear utility in the treatment of topically administered diseases associated with inflammation as noted herein such as, but not limited to, inflammatory bowel disease, contact dermatoses, actinic keratosis, psoriasis, or conjunctivitis.
• [-1H] a molecule that contains tritium atoms, a radioactive isotope
• A23187 a compound that allows free entry of calcium into a cell
• AA arachidonic acid, 5-8-11-14 eicosatetraenoic acid
• arachidonate arachidonic acid contained within a phospholipid
• free arachidonic acid arachidonic acid that is not contained within a phospholipid
• [ 2 H8]arachidonic acid the form of arachidonic acid labeled with 8 deuterium atoms, a stable isotope
• BSA bovine serum albumin
• CoA coenzyme A
• CoA-IT CoA-independent transacylase
• COX cyclooxygenase
• DTT dithiothreitol
• COX cyclooxygenase
• DTT dithi

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